Manufacture of 21 hydroxy steroids



MANUFACTURE OF 21 HYDROXY STEROIDS Albert Wettstein and Ernst Vischer,Basel, and Charles Meystre, Arlesheim, Switzerland, assignors to CibaPharmaceutical Products Inc., Summit, N. J.

No Drawing. Application June 29, 1955,

Serial No. 518,922

11 Claims. (Cl. 195-51) This invention relates to the manufacture ofsteroids by a process in which oxygen is introduced into steroidcompounds.

It is already known to introduce hydroxyl groups into steroids, forexample into the 21-position thereof, by the use of enzymes fromsuprarenal glands, especially using homogenates or by perfusion of theintact glands. However interesting this function of animal enzymes maybe from a theoretical point of view, it has not been capable ofsatisfactory practical application for the preparative, and especiallyindustrial production of ZI-hydroxy steroids. By the use ofmicrobiological methods, which are also applicable on a large scale, thespecified reaction has hitherto been incapable of execution.

We have now discovered that when compounds of the pregnane series, whichare unsubstituted in the 21- position, are oxygenated by contact withenzymes produced by aerobic cultures of fungi of the species Ophiobolusherpotrichus or Sclerotinia fructicola, there results a series ofsteroids which are hydroxylated in the ill-position. As startingmaterials for the new process one may suitably employ those compounds ofthe pregnane series which are unsubstituted in 2l-position, among whichare the saturated and unsaturated, unsubstituted or substitutedderivatives of any configuration of 10,13 dimethyl 17ethyl-cyclopentano-polyhydrophenanthrene, and also of its higher andlower homologues, for example corresponding A-nor, D-homoand19-norcompounds. Double bonds may occur, for example, in 1-, 4-, 5-, 6-,7-, 9-, 11-, 14-, 15- and/or l6-position. The preferred startingmaterials are those whose configuration is that of pregnane,Soc-pregnane, 17a-pregnane or corresponding racemates, such as thoseobtained in total synthesis. As substituents these compounds may containfree hydroxyl, oxo or carboxyl groups or the corresponding ester, ether,thioester, thioether, thiol ester, =thione ester, acetal, mercaptal,ketal, hydrazone, semicarbazone and cool groups, for example in 2-, 3-,6-, 7-, 11-, 12-, 16-, 17-, 18-, 19- and 20-position, as well as halogenatoms, such as for example chlorine or fluorine, e. g. in 9- and17-positions. As specific examples of preferred starting materials maybe given progesterone, 17 a progesterone, 16a hydroxy-progesterone,17u-hydroxy-progesterone, ll-keto-progesterone, Ila-and 11 8-hydroxy-progesterone, 9,11- or 11,12-dehydro-progesterone,19-oxo-progesterone, 1l-keto-l7a-hydroxy-progesterone, llocand1lfi-hydroxy-17a-hydroxy-progesterone, 9-ch1oroor9-fiuoro-1118,17a-dihydroxy-progesterone, 1 113, 18-dihydroxy-progesterone, 113,17,18-trihydroxy-progesterone,1l/s-hydroxy-l8-oxo-progesterone, 9-chloroor9-fluoro-1IB-hydroxy-l8-oxo-progesterone, 11,18 dioxoprogesterone,19-nor-progesterone, 19-nor-11B-hydroxy- 18-oxo-progesterone,pregnenolone, the corresponding 1- dehydro compounds, e. g.l-dehydro-progesterone, 1- dehydro 17a hydroxy-progesterone,1-dehydro-17a-hydroxy-ll-ketone-, and -11aor -11fi-hydroxy-progesterone.

The enzymes used are of microbiological origin and ted States Patent G"ice 2,778,776 Patented Jan. 22, 1957 produced by aerobic cultures offungi of the species Ophiobolus herpotrichus or Sclerotinia fructicola,although other known species of the aforementioned genera may also besuitably employed. For the introduction of oxygen, the startingmaterials are incubated at a pH between 3 and 8, in most'cases directly,with the submerged growth cultures of the specified fungi obtained underknown aerobic conditions. These cultures are advantageously agitated, i.e. shaken or stirred. A preferred medium is one which contains a sourceof assimilable carbon, especially carbohydrates, such as glucose,lactose, glycerol, mannose, and also, if desired, growth promotingsubstances, for example corn steep liquor or beer worts, and inorganicsalts, such as nitrates, chlorides, phosphates, tartrates of sodium,potassium or calcium and the corresponding ammonium salts. Thus natural,synthetic or semisynthetic nutrient solutions can be used.

In a preferred embodiment of the process of this invention, a volume ofnutrient medium containing assimilable carbohydrates and inorganic saltsis inoculated with a culture of a fungus of the species Ophz'obolusherpetrichus or Scleroti-nia fructicala. After a 3 to 5 day period ofincubation at a temperature of from about 20 C. to 35 C., a quantity ofa pregnane compound unsubstituted in the 2l-position is added to theculture medium, preferably in the form of a fine dispersion or asolution in an organic solvent, such as methanol, acetone or ethyleneglycol. The mixture is then further incubated for an additional periodof from 1 to 5 days, preferably about 3 to 4 days, at the sametemperature. The liquor is then separated from the mycelium by aconvenient means, such as filtration and the filtrate is extracted witha suitable organic solvent in which the newly formed 2l-oxygenatedsteroid is soluble, such as, for example, ethyl acetate, butyl acetate,methylene chloride, or chloroform. If desired, both the filtrate and themycelium mass may be extracted in order to insure complete removal ofthe product. The extract is Washed, dried and evaporated, and the21-oxygenated steroid is separated in pure form by suitable means, suchas adsorption on diatomaceous earth followed by elution with organicsolvents, as for example chloroform, acetone and mixtures thereof.Alternatively, the product may be purified by adsorption chromatographyemploying aluminum oxide as the matrix and mixtures of benzene, etherand ethyl acetate as solvents. Another method of purification, which maybe advantageously employed, is crystallization from organic solvents,such as acetone, ether, methylene chloride and mixtures thereof. Ifdesired, purification may be accomplished by conversion into functionalderivatives, such as Girard compounds. It is to be noted further thatthe process of this invention can also be advantageously carried out byfirst separating the active enzymes from corresponding aerobic cultures,for example, by extraction of the well developed mycelium, and usingthem with the exclusion of the growing cultures.

The products resulting from the process of this invention are known,therapeutically useful compounds. Some of the more notable examples ofcompounds which may be prepared in accordance with the process of thisinvention are cortisone, hydro-cortisone, l-dehydro-cortisone,1-dehydro-hydro-cortisone, Reichsteins substance S and aldosterone. Allof these compounds have established utility as therapeutic agents,particularly in the treatment of such diseases as arthritis and Addisonsdisease.

The following examples illustrate the invention:

Example 1 4 liters of 70 percent beer wort are sterilized in a shakingvessel (pH 5.6) and inoculated with a culture of Oplziobolusherpotrichus. .After 3 days shaking a arrears 3 26 C., there is addedunder sterile conditions to the well-developed culture a solution "of1.0 gram of progesterone in 25 cc. of acetone and shaking is continuedat the same temperature. After 4 days the mycelium is separated. Theculture filtrate is extracted three times, in each case with 1 liter ofethyl acetate and the combined extracts are washed with N-hydrochloricacid, N-sodium bicarbonate solution and water. The ethyl acetatesolution, dried over sodium sulfate, is evaporated under vacuum at 40 C.The residue (1.2 grams) is chromatographed on grams of silica gel byelution first with methylene chloride, then with chloroform and finallywith chloroform-acetone mixtures of increasing acetone content. Theindividual fractions (each 100 cc.) are evaporated and investigated bypaper chromatography. The methylene chloride fractions and the firstfractions eluted with chloroform contain together with impurities onlystarting material, whereas in the last chloroform fractions, and also inthe chloroform-acetone eluates (95:5 and 90:10) cortexone(ll-desoxy-corticosterone) is found to be present. The latter fractionsare combined and evaporated. The residue, which consists for the mostpart of cortexone (0.81 gram) is recrystallized from ether, wherebycolorless plates of M. P. 140-142 C. are obtained. These crystalspossess an optical rotation of +175 and in admixture with an authenticsample show no reduction of the melting point. Their infra-red andultra-violet spectra are also identical with those of cortexone.

Example 2 A 150 cc. quantity of a nutrient solution containing per liter50 grams of crude glucose, cc. of corn steep liquor and 10 grams ofammonium tartrate and the remainder tap water, is sterilized in a flaskof 500 cc. capacity and inoculated with a culture of Ophioboluslierpotrichus. After 2 days shaking at 26 C., the culture becomes welldeveloped and there is'then added under sterile conditions a solution of30 mg. of ll-kcto-progeschromatographic purification on a silicagel'column.

Example 3 To a culture of Ophiabolus herpotrichus such as that describedin Example 2, there is added a solution of 30 mg. of17a-hydroxy-progesterone, in 1.5 cc. of acetone. After incubation andextraction analogous to that set forth in Example 2, there is obtained acrude reaction product which consists for the most part of17oz-i1Yd1'OXY- cortexone (Reichsteins substance S). This can beobtained in crystalline form'by chromatographic purification on a silicagel column.

Example 4 A solution of 1 gram of l-dehydro-progesterone in cc. ofacetone is added under sterile conditions to 4 liters of an agitatedculture of Ophiobolus herpotrichus, obtained as described in Example 1.The mixture is agitated for 3 days at 27 C. and the mycelium isseparated. The culture filtrate is extracted several times with a totalof 3 liters of ethyl acetate. The combined extracts are washed with 1/ION-hydrochloric acid, lN-sodium bicarbonate solution and water. Theethyl acetate solution is dried over sodium sulfate and evaporated underreduced pressure, and the residue obtained (1.1 gram) is chromatographedon 30 grams of silica gel, elution'being carried out with chloroform andmixtures of chloroform and Example 5 A solution of 1 gram ofl-dehydro-l1-keto-17a-hy- (irony-progesterone in 25 cc. of acetone isadded to a culture of Oplziobolits herpotrichus as set forth in Example4 and the mixture is worked up and chromatographed as described in saidexample; The resultant chloroformacetone (1 :1) fractions consistchiefly of l-dehydrocortisone, which crystallizes from mixtures ofacetone and ether. The melting point is at 231-233 C.

l-dehydro-l1keto17u hydroxy-progesterone can prepared for example asfollows: To a solution of 5 grams of3,11,20-trilteto-l7a-hydroxy-allopregnane in 50 cc. of acetic 'acidecontaining a few drops of a concentrated solution of hydrogen bromide inacetic acid there are added cc. of acetic acid containing 1 mol ofbromine. After discoloration the second mol of bromine in 80 cc. ofacetic acid is added and the mixture is stirred for 1 hour at roomtemperature. The reaction product is obtained by evaporating the solventin vacuo, dissolving the residue in methylene chloride, washing themethylene chloride solution with water, dilute sodium bicarbonatesolution and again with water and evaporating the solvent in vacuo. 5grams of the so-obtained crude 2,4-dibromo-3 ,11,ZO-triketo-l7oz-hydroxy-allopregname are heated with 20 cc. ofcollidine to about C. After cooling, water is added to the reactionmixture and the reaction product is extracted with methylene chloride.The methylene chloride solution is washed with dilute sulfuric "acid andwatendried and evaporated, wherebyl-dehydro-ll-keto-17a-hydroXy-progesterone is obtained.

Examplefi Example 7 A 50 cc. quantity of beerwortof 70 percent strengthis sterilized in a flask and inoculated with a culture of Sclerotiniafruc tieola. ,The culture is agitated at 27 C. for 4 days. Thereis thenadded, under sterile conditions, a solution of 10 mg. of progesterone in0.5 cc. of acetone. The mixture is agitated for another 4 days at thesame temperature, and the mycelium then sep' arated. The culturefiltrate is exhaustively extracted with ethyl acetate. The extract iswashed with water, dried, -and evaporated under reduced pressure.Paperchromatographic investigation of the resulting crudeextractshowsthat it consistsfor the most part of cortexonc 11-desoxy-ccrticosterone).

Example 8 A solution of 10 mg. ofl-dehydro-llketo-l7a-hydroxy-progesterone in 0.5 cc. of acetone isaddedto a culture of Scleratinia j ructicola asset forthin Example 7 andthe mixture is worked up and chromatographed as described in saidexample. The crude extract consists mainly of l-dehydro-cortisone.

Example 9 A 120 cc. quantity of beer wort is sterilized in a flask of500 cc. capacity and inoculated with Ophiobolus herpotrichus. Theculture is agitated for 4 days at 26 C. A solution of 30 mg. of d,1-A-3:ZO-diketo-llfl-hydroxypregnene-l8-acid-lactone-(18 115) in 1.5 cc. ofacetone is then added to the culture under sterile conditions and themixture is agitated for an additional 3 days at 26 C. The culture isextracted with ethyl acetate as described inthe preceding examples.The-crude extract is worked up by a preparative paper chromatography(system propyleneglycol-toluene), a substance being separated ofi whichis more highly polar than the starting material and reduces alkalinesilver diamine solution rapidly and strongly. It is crystallized from amixture of acetone and ether and is d,l-A-3:20-diketo-l15:21-dihydroxypregnene-l8-acid-lactone-(18- 115).

The d,1-A -3 :20-diketo-1l/i-hydroxy-pregnene-18-acid- 1actone-(18- 11p)can be prepared in accordance with the procedure set forth in detail incopending U. S. application Serial Number 480,062 filed January 5, 1955by Tadeus Reichstein et al. In general, it may be stated that thiscompound is obtained by hydrogenating d,1- A5116 3 ethylenedioxy 20 keto115 hydroxypregnadiene-18-acid-lactone-(18 116) in ethanol solution inthe presence of a palladium strontium carbonate catalyst and splittingthe ketal group in 3-position with p-toluene-sulfonic acid in acetonesoluton to form d,1- A 3:20 diketo 11/3 hydroxy pregnene 18acidlactone-(18 11,8).

What is claimed is:

1. In a process for the production of 21-hydr0xy1ated pregnanecompounds, the step which comprises subjecting a compound of thepregnane series, in which compound the 21-position is unsubstituted, tothe action of enzymes produced by aerobically cultivating a fungusselected from the group consisting of the species Ophiobolusherpotrichus and Sclerotinia fructicola, and separating the resultant21-hydroxylated steroid.

2. In a process for the production of 21-hydroxylated pregnanecompounds, the step which comprises subject:

ing a A -pregnadiene compound, in which compound the 21-position isunsubstituted, to the action of enzymes produced by aerobicallycultivating a fungus selected from the group consisting of the speciesOphiabolus herpotrichus and Sclerotinia fructicola and separating theresulting 21-hydroxylated steroid.

3. In a process for the production of 21-hydroxylated pregnanecompounds, the step which comprises subjecting a compound of thepregnane series, in which compound the 21-position is unsubstituted,under aerobic conditions to the action of a growing fungus selected fromthe group consisting of the species Ophiobolus herpotrichus andSclerotinia fructicola.

4. In a process for the production of 21-hydroxylated pregnanecompounds, the step which comprises subjecting a compound of thepregnane series, in which compound the 21-position is unsubstituted,under aerobic conditions to the action of enzymes produced by aerobiccultivation of a fungus selected from the group consisting of thespecies Ophiobolus herpotrichus and Sclerotinia fructicola.

5. In a process for the production of A -3,11,20-trioxo-ZI-hydroXy-pregnene the step which comprises subjectingll-keto-progesterone to the action of a growing culture of a fungus ofthe species Ophiobolus herpotrichus and separating the so formeddehydro-corticosterone.

6. A process as set forth in claim 1, wherein the pregnane compound isprogesterone.

7. A process as set forth in claim 1, wherein the pregnane compound isll-keto-progesterone.

8. A process as set forth in claim 1, wherein the pregnane compound is17a-hydroxy-progesterone.

9. A process as set forth in claim 2, wherein the starting material isl-dehydro-progesterone.

10. A process as set forth in claim 2, wherein the starting material isl-dehydro-l1-keto-17u-hydroxy-progesterone.

11. A process as set forth in claim 2, wherein the starting material isl-dehydro-l1,8,17a-dihydroxy-progesterone.

References Cited in the file of this patent Mey stre et a1.: HelveticaChimica Acta, 37,1954, pages 1548-1553.

1. IN A PROCESS FOR THE PRODUCTION OD 21-HYDROXYLATED PERGNANECOMPOUNDS, THE STEP WHICH COMPRISES SUBJECTING A COMPOUND OF THEPREGNANE SERIES, IN WHICH COMPOUND THE 21 POSITION IS UNSUBSTITUTED, TOTHE ACTION OF ENZYMES PRODUCED BY AEROBICALLY CULTIVATING A FUNGUSSELECTED FROM THE GROUP CONSISTING OF THE OPHIOBOLUS HERPOTRICHUS ANDSCLEROTINIA FRUCTICOLA, AND SEPARATING THE RESULTANT 21-HYDROXYLATEDSTERIOD.